Targeting receptor kinases by a novel indolinone derivative in multiple myeloma: abrogation of stroma-derived interleukin-6 secretion and induction of apoptosis in cytogenetically defined subgroups by Guido Bisping, Martin Kropff, Doris Wenning, Britta Dreyer, Sergey Bessonov, Frank Hilberg, Gerald J. Roth, Gerd Munzert, Martin Stefanic, Matthias Stelljes, Christian Scheffold, Carsten Müller-Tidow, Peter Liebisch, Nicola Lang, Jöelle Tchinda, Hubert L. Serve, Rolf M. Mesters, Wolfgang E. Berdel, and Joachim Kienast Blood Volume 107(5):2079-2089 March 1, 2006 ©2006 by American Society of Hematology

Inhibition of VEGFR2 (KDR) and FGFR3 phosphorylation by BIBF 1000. Inhibition of VEGFR2 (KDR) and FGFR3 phosphorylation by BIBF 1000. (A) Cultured marrow stroma cells were exposed to VEGF (50 ng/mL) in the presence of increasing concentrations of BIBF 1000 for 5 minutes. Dose-dependent abrogation of VEGFR2 phosphorylation (top: anti–p-Tyr100 immunoblots, densitometry) and corresponding immunoblots of total VEGFR2 (bottom) are shown. (B) OPM-2 cells known to express a constitutively active FGFR3 mutant were incubated with increasing concentrations of BIBF 1000 for 5 minutes. FGFR3 phosphorylation was inhibited in a dose-dependent manner (top: anti–p-Tyr85 immunoblots, densitometry). Bottom panel shows corresponding immunoblots of total FGFR3. Guido Bisping et al. Blood 2006;107:2079-2089 ©2006 by American Society of Hematology

Effects of BIBF 1000 on stroma-derived interleukin-6 (IL-6) release and related inhibition of MAPK phosphorylation. Effects of BIBF 1000 on stroma-derived interleukin-6 (IL-6) release and related inhibition of MAPK phosphorylation. (A-B) Effects of BIBF 1000 on bone marrow stroma cell (BMSC)–derived IL-6 release: BIBF 1000 (0.5 μM) significantly inhibited basal IL-6 release from BMSCs and IL-6 secretion induced by VEGF121, VEGF165, bFGF, and TGF-β (105.3 ± 28.1 versus 37.3 ± 16.1 [vehicle control versus BIBF 1000]; 169.2 ± 23.4 versus 45.3 ± 16.6 [VEGF121 versus VEGF121 + BIBF 1000]; 172.0 ± 22.3 versus 60.1 ± 21.9 [VEGF165 versus VEGF165 + BIBF 1000]; 185.1 ± 17.3 versus 44.7 ± 14.3 [bFGF versus bFGF + BIBF 1000]; 499.5 ± 33.8 versus 195.7 ± 36.7 3 [TGF-β versus TGF-β + BIBF 1000], respectively; P < .001). In contrast, IL-6 release on exposure to TNF-α or IL-1β was not significantly reduced (754.7 ± 122.6 versus 547.4 ± 83.0 [TNF-α versus TNF-α + BIBF 1000]; 1881.7 ± 122.3 versus 1855.8 ± 122.9 [IL-1β versus IL-1β + BIBF 1000]). IL-6 concentrations were determined in triplicates from supernatants of BMSC cultures derived from different patients with myeloma (n = 4). BMSCs were starved prior to incubation with either VEGF121, VEGF165, bFGF, TGF-β, TNF-α, or IL-1β ± BIBF 1000 and kept in serum-free conditions for 72 hours. Data are presented as means ± SE. The Wilcoxon test was used to identify differences between controls and corresponding BIBF 1000–treated BMSCs (means ± SE). (C) Inhibition of MAPK phosphorylation by BIBF 1000 in BMSCs: subconfluent BMSCs were starved for 4 hours in serum-free medium and subsequently exposed for 2 hours to either VEGF (50 ng/mL), bFGF (10 ng/mL), TGF-β (10 ng/mL), TNF-α (10 ng/mL) or IL-1β (10 ng/mL) in the absence or presence of BIBF 1000 (1, 5, or 10 μM), to BIBF alone (10 μM) or to the MAPK-inhibitor PD98059 (20 μM). Proteins were extracted, boiled, and separated by SDS-electrophoresis. Subsequent immunoblotting was performed using a rabbit polyclonal p-MAPK (p44/42) antibody and a corresponding goat anti–rabbit HRP–conjugated secondary antibody. Notably, inhibition of MAPK phosphorylation by BIBF 1000 was most effective in BMSCs exposed to VEGF, bFGF, or TGF-β. The results shown are representative of 3 independent experiments. Guido Bisping et al. Blood 2006;107:2079-2089 ©2006 by American Society of Hematology

IL-6 secretion in transwell and contact cocultures of BMSCs and MM cells is significantly inhibited by BIBF 1000. IL-6 secretion in transwell and contact cocultures of BMSCs and MM cells is significantly inhibited by BIBF 1000. (A) Control experiments in BMSC monocultures showed dose-dependent inhibition of IL-6 release by BIBF 1000 (0.5 μM). (B) Basal IL-6 secretion by myeloma cell lines ranged near or below the detection limit of the assay. (C-H) As compared with BMSC monocultures, IL-6 secretion was increased in both transwell (C-E) and contact (F-H) cocultures of BMSCs with U-266, RPMI-8226, or KMS-11 cells. Exposure to BIBF 1000 (0.125-1.0 μM) resulted in almost complete abrogation of IL-6 secretion in both types of cocultures. (I-J) In transwell cocultures of BMSCs and patient-derived CD138+-sorted MM cells, a similar increase in IL-6 secretion as compared with the respective monocultures and its dose-dependent inhibition by BIBF 1000 was observed. IL-6 concentrations were determined in serum-free supernatants of 72-hour cultures. Samples were measured in triplicates, and the results are presented as means ± SEs of at least 3 independent experiments. Significance of group differences was analyzed by the Mann-Whitney rank sum test. *P < .05, **P < .005, ***P < .001 versus controls without BIBF 1000. Guido Bisping et al. Blood 2006;107:2079-2089 ©2006 by American Society of Hematology

Inhibition of myeloma cell adhesion to BMSCs by BIBF 1000. Inhibition of myeloma cell adhesion to BMSCs by BIBF 1000. Contact cocultures were performed with confluent monolayers of 1 × 105 BMSCs and 2 × 106 CFSE-prelabeled RPMI-8226, U-266, OPM-2, or KMS-11 cells, respectively. Cell cultures were incubated in the absence or in the presence of BIBF 1000 (0.25 to 5.0 μM) for 24 hours. Subsequently, nonadherent cells were washed off 3 times. The remaining adherent cells were trypsinized and quantified by flow cytometry. Data are presented as mean ratios of adherent MM cells over BMSCs from 3 independent experiments for each cell line. Analysis of significance for overall group differences was performed by the Kruskal-Wallis test (U-266, OPM-2, and KMS-11: P = .001; RPMI-8226: NS). Guido Bisping et al. Blood 2006;107:2079-2089 ©2006 by American Society of Hematology

Dose-dependent inhibition of myeloma cell proliferation by BIBF 1000. Dose-dependent inhibition of myeloma cell proliferation by BIBF 1000. (A-C) Myeloma cells (5 × 104) per well were seeded onto 96-well plates and cultured in RPMI-1640 medium with or without BIBF 1000. Cells were pulsed with 0.625 μCi (0.023 MBq) 3H-thymidine per well for 72 hours. Cells were harvested, and 3H-thymidine incorporation was counted. 3H-thymidine uptake is shown as mean values of triplicate determinations from 2 independent series of experiments for each cell line. 3H-thymidine uptake was normalized to the controls without BIBF 1000. A 70% or greater inhibition of proliferation by 1.0 μM BIBF 1000 was observed in the t(4;14)–positive KMS-11, OPM-2, and NCI-H929 cells and in the t(14;16)–positive MM.1S and MM.1R cells (P < .001; BIBF 1000 [1.0 μM] versus control without BIBF 1000). BIBF 1000 (1 μM) inhibited the proliferation of U-266 and L-363 by approximately 50%, whereas it did not or only marginally influence the proliferation of the t(14;16)–positive cell lines JJN-3 and ANBL-6 as well as of the t(16;22)–positive cell line RPMI-8226. Guido Bisping et al. Blood 2006;107:2079-2089 ©2006 by American Society of Hematology

Induction of apoptosis by BIBF 1000 in patient myeloma cells. Induction of apoptosis by BIBF 1000 in patient myeloma cells. Quantification of apoptosis by flow cytometry in CD138+-sorted cells from the marrow of a patient with t(4;14)–positive myeloma, [pt 3]). Increased early/late apoptosis by BIBF 1000, dexamethasone, and their combination is depicted in the top row. The bottom row shows partial reversal of apoptosis by IL-6. Percentage quantification of early apoptotic cells (annexin V–positive and propidium iodide [PI]–negative; bottom right quadrants) or late apoptotic cells (annexin V–positive and PI-positive; top right quadrants) as indicated in dot plot panels. Guido Bisping et al. Blood 2006;107:2079-2089 ©2006 by American Society of Hematology

BIBF 1000–induced apoptosis in t(4;14)–positive myeloma cells and related inhibition of MAPK (p44/42) phosphorylation. BIBF 1000–induced apoptosis in t(4;14)–positive myeloma cells and related inhibition of MAPK (p44/42) phosphorylation. (A) Dose-dependent induction of apoptosis in t(4;14)–positive, FGFR3-overexpressing OPM-2 cells by BIBF 1000 (125-500 nM, top). Induction of apoptosis was completely reverted by the pan-caspase-inhibitor z-VAD-FMK (100 μM). No significant BIBF 1000-induced apoptosis occurred in U-266 cells serving as a t(4;14)–negative control (bottom). (B) FGFR3 protein expression in t(4;14)–positive OPM-2 and KMS-11 versus t(4;14)–negative RPMI-8226 and U-266 cells. Representative immunoblots are shown. (C) Inhibition of MAPK phosphorylation in t(4;14) OPM-2 cells. Constitutive and IL-6–induced MAPK phosphorylation were almost completely or partially inhibited by BIBF 1000, whereas STAT3 phosphorylation was not affected. (D) Inhibition of MAPK phosphorylation in t(4;14) KMS-11 cells. Results were similar to those obtained with OPM-2 cells. (E-F) Neither MAPK nor STAT3 phosphorylation were altered by BIBF 1000 in N-Ras mutated t(4;14)–positive NCI-H929 cells (E) and in t(4;14)–negative U-266 cells (F). In panels C through F, myeloma cell lines were starved for 2 hours and exposed to IL-6 (10 ng/mL) and/or BIBF 1000 (500 nM) for 5 minutes prior to extraction of proteins by RIPA-buffer containing protease and phosphatase inhibitors. Guido Bisping et al. Blood 2006;107:2079-2089 ©2006 by American Society of Hematology

BIBF 1000 induced apoptosis in t(14;16)–positive MM BIBF 1000 induced apoptosis in t(14;16)–positive MM.1S cells and related inhibition of the phosphatidyl-inositol-3 kinase (PI3-K)/AKT pathway. BIBF 1000 induced apoptosis in t(14;16)–positive MM.1S cells and related inhibition of the phosphatidyl-inositol-3 kinase (PI3-K)/AKT pathway. (A) BIBF 1000 induced apoptosis in native t(14;16)–positive MM.1S cells and had additive apoptotic effects in the presence of Dex (compare percentages of annexin V-positive, PI-negative cells, lower right quadrants). Apoptosis induced by BIBF 1000 was partially antagonized by IL-6 at saturating concentrations (10 ng/mL) and completely prevented by the pan-caspase inhibitor z-VAD. Percentage quantification of early apoptotic cells (annexin V–positive and PI-negative; bottom right quadrants) or late apoptotic cells (annexin V–positive and PI-positive; top right quadrants) as indicated in dot plot panels. (B) BIBF 1000 had no effect on MAPK or STAT3 phosphorylation in MM.1S cells. (C) The PI3-K/AKT pathway was inhibited by BIBF 1000 both in the absence and presence of IL-6 as shown by decreased phosphorylation of AKT. (D) BIBF 1000 (0.5 μM) and the PI3-K inhibitor Ly294002 (10 μM) had similar effects on the induction of early and late apoptosis in MM.1S cells (annexin V–positive [+ve], PI-negative [-ve], and annexin V–positive, PI-positive cells, respectively; means ± SE). Guido Bisping et al. Blood 2006;107:2079-2089 ©2006 by American Society of Hematology